Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.444
Filtrar
1.
Microbes Environ ; 35(1)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32037377

RESUMO

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.


Assuntos
Genoma Bacteriano/genética , Elementos da Série dos Lantanídeos/metabolismo , Proteobactérias/genética , Proteobactérias/isolamento & purificação , Oxirredutases do Álcool/genética , Proteínas de Bactérias/genética , Meios de Cultura/metabolismo , Sedimentos Geológicos/microbiologia , Metais Terras Raras/metabolismo , Metano/metabolismo , Methylosinus/classificação , Methylosinus/genética , Methylosinus/isolamento & purificação , Methylosinus/metabolismo , Tanques/microbiologia , Proteobactérias/classificação , Proteobactérias/fisiologia , RNA Ribossômico 16S/genética
2.
Proc Natl Acad Sci U S A ; 117(3): 1485-1495, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31911473

RESUMO

Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins' sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins' sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily.


Assuntos
Proteínas de Escherichia coli/química , Proteínas Intrinsicamente Desordenadas/química , Dobramento de Proteína , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Simulação de Dinâmica Molecular , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/metabolismo , Biossíntese de Proteínas , Metiltransferases de Proteína/química , Metiltransferases de Proteína/genética , Metiltransferases de Proteína/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
3.
Appl Biochem Biotechnol ; 190(1): 18-29, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31301008

RESUMO

NAD(P)H-dependent enzymes are ideal biocatalysts for the industrial production of chiral compounds, such as chiral alcohols, chiral amino acids, and chiral amines; however, efficient strategies for the regeneration of coenzyme are expected as costly of the coenzymes. Herein, a solvent-tolerant isopropanol dehydrogenase (IDH) showing lower similarity (37%) with other proteins was obtained and characterized. The enzyme exhibits high catalysis ability of its substrates methanol, ethanol, ethylene glycol, glycerol, isopropanol, n-butanol, isobutanol, and acetone. And it has good adaptability in organic solvents (isopropanol, acetonitrile, acetone, and acetophenone). Interaction force and the corresponding amino acid residues between IDH and NAD+ or NADP+ were parsed by docking. The wide substrate spectrum, excellent organic solvent tolerance, and good biocatalytic activity make the excavated enzyme a promising biocatalyst for the production of chiral compounds industrially and the construction of coenzyme regeneration systems in aqueous organic phase or organic phase.


Assuntos
Oxirredutases do Álcool/metabolismo , Coenzimas/metabolismo , Solventes/metabolismo , Oxirredutases do Álcool/genética , Sítios de Ligação , Clonagem Molecular , Cinética , Simulação de Acoplamento Molecular , NAD/metabolismo , NADP/metabolismo , Compostos Orgânicos/metabolismo , Especificidade por Substrato
4.
J Chem Ecol ; 46(2): 217-231, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31879865

RESUMO

Despite active research, antiherbivore activity of specific plant phenolics remains largely unresolved. We constructed silver birch (Betula pendula) lines with modified phenolic metabolism to study the effects of foliar flavonoids and condensed tannins on consumption and growth of larvae of a generalist herbivore, the autumnal moth (Epirrita autumnata). We conducted a feeding experiment using birch lines in which expression of dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS) or anthocyanidin reductase (ANR) had been decreased by RNA interference. Modification-specific effects on plant phenolics, nutrients and phenotype, and on larval consumption and growth were analyzed using uni- and multivariate methods. Inhibiting DFR expression increased the concentration of flavonoids at the expense of condensed tannins, and silencing DFR and ANR decreased leaf and plant size. E. autumnata larvae consumed on average 82% less of DFRi plants than of unmodified controls, suggesting that flavonoids or glandular trichomes deter larval feeding. However, larval growth efficiency was highest on low-tannin DFRi plants, indicating that condensed tannins (or their monomers) are physiologically more harmful than non-tannin flavonoids for E. autumnata larvae. Our results show that genetic manipulation of the flavonoid pathway in plants can effectively be used to produce altered phenolic profiles required for elucidating the roles of low-molecular weight phenolics and condensed tannins in plant-herbivore relationships, and suggest that phenolic secondary metabolites participate in regulation of plant growth.


Assuntos
Betula/química , Flavonoides/metabolismo , Mariposas/fisiologia , Plantas Geneticamente Modificadas/química , Taninos/metabolismo , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Betula/enzimologia , Betula/parasitologia , Flavonoides/farmacologia , Herbivoria/efeitos dos fármacos , Interações Hospedeiro-Parasita , Larva/crescimento & desenvolvimento , Larva/fisiologia , Mariposas/crescimento & desenvolvimento , Oxigenases/antagonistas & inibidores , Oxigenases/genética , Oxigenases/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Interferência de RNA , Taninos/farmacologia
5.
Support Care Cancer ; 28(1): 395-403, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31056713

RESUMO

PURPOSE: Asthenia, myalgia, arthralgia, mucositis, abdominal pain, diarrhea, and neutropenia are adverse reactions commonly reported by women undergoing chemotherapy. Traditional approaches do not take into account the effect that chemotherapeutic changes and variable interactions can cause in adverse reactions. We aimed to identify the impact of the change of a chemotherapy protocol within the same treatment in profiles associated with adverse reactions. METHODS: A total of 166 women admitted to the Brazilian National Institute of Cancer (INCA) were followed. Polymorphisms, clinical variables, and FAC-D protocols (3 cycles of cyclophosphamide, 5-fluorouracil, and doxorubicin followed by 3 cycles of docetaxel) composed the set of independent variables analyzed. Reaction levels were recorded at the end of each chemotherapy cycle via interviews. Marginal models were fitted. RESULTS: The results of marginal models for non-hematological reactions revealed that the docetaxel phase was associated with increased reaction levels compared with the FAC phase. In addition, the set of factors associated with the reactions changed in each protocol. The post-menopausal status was related to high levels of asthenia in docetaxel protocol whereas CYP2B6 polymorphism (rs3745274) was related to high levels in FAC protocol. Regarding the docetaxel phase, high levels of abdominal pain and mucositis were related to CBR3 gene (rs8133052) polymorphism and diabetes respectively. CONCLUSION: The results suggest the need for monitoring non-hematological reactions during the docetaxel phase of FAC-D treatment. The factors related to more severe reactions depend on the chemotherapy protocol used.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Docetaxel/administração & dosagem , Substituição de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Dor Abdominal/induzido quimicamente , Dor Abdominal/epidemiologia , Dor Abdominal/genética , Adulto , Idoso , Oxirredutases do Álcool/genética , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Brasil/epidemiologia , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Estudos de Coortes , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Citocromo P-450 CYP2B6/genética , Diarreia/induzido quimicamente , Diarreia/epidemiologia , Diarreia/genética , Docetaxel/efeitos adversos , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Substituição de Medicamentos/métodos , Substituição de Medicamentos/estatística & dados numéricos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/tratamento farmacológico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Predisposição Genética para Doença , Humanos , Estudos Longitudinais , Pessoa de Meia-Idade , Testes Farmacogenômicos , Polimorfismo de Nucleotídeo Único
6.
PLoS One ; 14(12): e0227225, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31877197

RESUMO

One identified dihydroflavonol 4-reductases (DFR) encoding gene (named as CsDFRa herein) and five putative DFRs (named as CsDFRb1, CsDFRb2, CsDFRb3, CsDFRc and CsDFRd) in tea (Camellia sinensis) have been widely discussed in recent papers concerning multi-omics data. However, except for CsDFRa, their function and biochemical characteristics are not clear. This study aims to compare all putative CsDFRs and preliminarily evaluate their function. We investigated the sequences of genes (coding and promoter regions) and predicted structures of proteins encoded, and determined the activities of heterologously expressed CsDFRs under various conditions. The results showed that the sequences of five putative CsDFRs were quite different from CsDFRa, and had lower expression levels as well. The five putative CsDFRs could not catalyze three dihydroflavonol substrates. The functional CsDFRa had the strongest affinity with dihydroquercetin, and performed best at pH around 7 and 35°C but was not stable at lower pHs or higher temperatures. Single amino acid mutation at position 141 modified the preference of CsDFRa for dihydroquercetin and dihydromyricetin, and also weakened its stability. These data suggest that only CsDFRa works in the pathway for generating anthocyanidins and catechins. This study provides new insights into the function of CsDFRs and may assist to develop new strategies to manipulate the composition of tea flavonoids in the future.


Assuntos
Oxirredutases do Álcool/genética , Camellia sinensis/genética , Proteínas de Plantas/genética , Oxirredutases do Álcool/análise , Sequência de Aminoácidos , Camellia sinensis/química , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Família Multigênica , Filogenia , Proteínas de Plantas/análise , Alinhamento de Sequência
7.
Adv Exp Med Biol ; 1185: 209-213, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884613

RESUMO

Mutations in retinol dehydrogenase 12 (RDH12) cause a severe early-onset retinal degeneration, for which there is no treatment. RDH12 is involved in photoreceptor retinoid metabolism and is a potential target for gene therapy, which has been successful in treating RPE65-associated LCA. RDH12-associated retinal degeneration is particularly devastating due to early macular atrophy, which will likely impact therapeutic outcomes. Defining the unique features and natural history of disease associated with RDH12 mutations is a critical first step in developing treatments. The purpose of this review is to aggregate and summarize the body of literature on phenotypes in RDH12-associated retinal degeneration to help map the natural history of disease and identify phenotypic milestones in disease progression. The results reveal a severe blinding disorder with onset in early childhood and frequent retention of reduced yet useful vision until adolescence. The severity is associated with genotype in some cases. Distinct phenotypic features include macular atrophy followed by bone spicule pigment early in life, in contrast to other forms of LCA which often have a relatively normal fundus appearance in childhood despite severe visual dysfunction. Formal natural history studies are needed to define milestones in disease progression and identify appropriate outcome measures for future therapy trials.


Assuntos
Oxirredutases do Álcool/genética , Degeneração Retiniana/genética , Estudos de Associação Genética , Genótipo , Humanos , Mutação , Fenótipo
8.
Microb Cell Fact ; 18(1): 187, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675969

RESUMO

BACKGROUND: The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear. RESULTS: The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities. CONCLUSIONS: PAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression-heterologous genes included-, thus directly influencing the production kinetics of recombinant protein.


Assuntos
Oxirredutases do Álcool/genética , Proteínas Fúngicas/metabolismo , Metanol/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Dosagem de Genes , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Pichia/genética , Regiões Promotoras Genéticas
9.
Chem Commun (Camb) ; 55(96): 14462-14465, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31728457

RESUMO

Structure-based engineering of a NAD+-dependent secondary alcohol dehydrogenase from Micrococcus luteus led to a 1800-fold increase in catalytic efficiency for NADP+. Furthermore, the engineered enzymes (e.g., D37S/A38R/V39S/T15I) were successfully coupled to a NADPH-dependent Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440 for redox-neutral biotransformations of C18 fatty acids into C9 chemicals.


Assuntos
Oxirredutases do Álcool/metabolismo , Micrococcus luteus/enzimologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Sítios de Ligação , Biotransformação , Ácidos Graxos/metabolismo , Engenharia Genética , Cinética , Oxigenases de Função Mista/metabolismo , Simulação de Dinâmica Molecular , Oxirredução , Pseudomonas/enzimologia
10.
Acta Crystallogr D Struct Biol ; 75(Pt 10): 918-929, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31588923

RESUMO

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.


Assuntos
Oxirredutases do Álcool/química , Mononucleotídeo de Flavina/química , Actinobacteria/enzimologia , Oxirredutases do Álcool/genética , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Cinética , Mutação , Oxirredução , Especificidade por Substrato
11.
BMC Plant Biol ; 19(1): 434, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31638916

RESUMO

BACKGROUND: Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop's sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. RESULTS: To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40-1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40-1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. CONCLUSIONS: Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40-1 expression, whereas higher miR156 overexpression results in drought susceptibility.


Assuntos
Oxirredutases do Álcool/metabolismo , Medicago sativa/genética , MicroRNAs/genética , Oxirredutases do Álcool/genética , Secas , Regulação da Expressão Gênica de Plantas , Medicago sativa/enzimologia , Medicago sativa/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , RNA de Plantas/genética , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Nat Commun ; 10(1): 4068, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492851

RESUMO

The aldehyde dehydrogenase (ALDH) family of metabolic enzymes converts aldehydes to carboxylates. Here, we find that the reductive consequence of ALDH7A1 activity, which generates NADH (nicotinamide adenine dinucleotide, reduced form) from NAD, underlies how ALDH7A1 coordinates a broad inhibition of the intracellular transport pathways. Studying vesicle formation by the Coat Protein I (COPI) complex, we elucidate that NADH generated by ALDH7A1 targets Brefeldin-A ADP-Ribosylated Substrate (BARS) to inhibit COPI vesicle fission. Moreover, defining a physiologic role for the broad transport inhibition exerted by ALDH7A1, we find that it acts to reduce energy consumption during hypoxia and starvation to promote cellular energy homeostasis. These findings advance the understanding of intracellular transport by revealing how the coordination of multiple pathways can be achieved, and also defining circumstances when such coordination is needed, as well as uncovering an unexpected way that NADH acts in cellular energetics.


Assuntos
Oxirredutases do Álcool/metabolismo , Aldeído Desidrogenase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metabolismo Energético , Homeostase , Espaço Intracelular/metabolismo , Oxirredutases do Álcool/genética , Aldeído Desidrogenase/genética , Transporte Biológico , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Hipóxia Celular , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , NAD/metabolismo , Transdução de Sinais , Inanição
13.
Mol Cells ; 42(9): 672-685, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31486328

RESUMO

Currently, liver transplantation is the only available remedy for patients with end-stage liver disease. Conservation of transplanted liver graft is the most important issue as it directly related to patient survival. Carbonyl reductase 1 (CBR1) protects cells against oxidative stress and cell death by inactivating cellular membrane-derived lipid aldehydes. Ischemia-reperfusion (I/R) injury during living-donor liver transplantation is known to form reactive oxygen species. Thus, the objective of this study was to investigate whether CBR1 transcription might be increased during liver I/R injury and whether such increase might protect liver against I/R injury. Our results revealed that transcription factor Nrf2 could induce CBR1 transcription in liver of mice during I/R. Pre-treatment with sulforaphane, an activator of Nrf2, increased CBR1 expression, decreased liver enzymes such as aspartate aminotransferase and alanine transaminase, and reduced I/R-related pathological changes. Using oxygenglucose deprivation and recovery model of human normal liver cell line, it was found that oxidative stress markers and lipid peroxidation products were significantly lowered in cells overexpressing CBR1. Conversely, CBR1 knockdown cells expressed elevated levels of oxidative stress proteins compared to the parental cell line. We also observed that Nrf2 and CBR1 were overexpressed during liver transplantation in clinical samples. These results suggest that CBR1 expression during liver I/R injury is regulated by transcription factor Nrf2. In addition, CBR1 can reduce free radicals and prevent lipid peroxidation. Taken together, CBR1 induction might be a therapeutic strategy for relieving liver I/R injury during liver transplantation.


Assuntos
Carbonil Redutase (NADPH)/metabolismo , Transplante de Fígado , Fator 2 Relacionado a NF-E2/metabolismo , Traumatismo por Reperfusão/terapia , Regulação para Cima , Adulto , Oxirredutases do Álcool/genética , Animais , Biópsia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Glucose/deficiência , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/patologia , Doadores Vivos , Luteolina/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Oxigênio , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
14.
Int J Mol Sci ; 20(19)2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31554290

RESUMO

Grape hyacinth (Muscari spp.) is a popular ornamental plant with bulbous flowers noted for their rich blue color. Muscari species have been thought to accumulate delphinidin and cyanidin rather than pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. In our study, we clone a novel DFR gene from blue flowers of Muscari. aucheri. Quantitative real-time PCR (qRT-PCR) and anthocyanin analysis showed that the expression pattern of MaDFR had strong correlations with the accumulation of delphinidin, relatively weak correlations with cyanidin, and no correations with pelargonidin. However, in vitro enzymatic analysis revealed that the MaDFR enzyme can reduce all the three types of dihydroflavonols (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), although it most preferred dihydromyricetin as a substrate to produce leucodelphinidin, the precursor of blue-hued delphinidin. This indicated that there may be other functional genes responsible for the loss of red pelargonidin-based pigments in Muscari. To further verify the substrate-specific selection domains of MaDFR, an assay of amino acid substitutions was conducted. The activity of MaDFR was not affected whenever the N135 or E146 site was mutated. However, when both of them were mutated, the catalytic activity of MaDFR was lost completely. The results suggest that both the N135 and E146 sites are essential for the activity of MaDFR. Additionally, the heterologous expression of MaDFR in tobacco (Nicotiana tabacum) resulted in increasing anthocyanin accumulation, leading to a darker flower color, which suggested that MaDFR was involved in color development in flowers. In summary, MaDFR has a high preference for dihydromyricetin, and it could be a powerful candidate gene for genetic engineering for blue flower colour modification. Our results also make a valuable contribution to understanding the basis of color variation in the genus Muscari.


Assuntos
Oxirredutases do Álcool/genética , Antocianinas/biossíntese , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Vitis/genética , Vitis/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Flores/genética , Mutagênese Sítio-Dirigida , Fenótipo , Filogenia , Pigmentação , Desenvolvimento Vegetal/genética , Plantas Geneticamente Modificadas , Proteínas Recombinantes , Análise de Sequência de DNA
15.
Gene ; 716: 144024, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31390541

RESUMO

The young leaves generally accumulate a certain concentration anthocyanins in the dominant species of the subtropical forest, and the changes of anthocyanin synthesis-related enzyme genes expression levels had an important effect on the study photoprotection of anthocyanins in the young leaves of subtropical forests. The determination of anthocyanin synthesis-related enzyme gene sequences and the selection of appropriate reference genes provide a basis for analyzing the functional properties of anthocyanins. In this study, four dominant subtropical forest species (i.e., Schima superba, Castanopsis fissa, Acmena acuminatissima, Cryptocarya concinna) were taken as materials. To obtain the correct nucleotide sequences of anthocyanin-related enzymes, the nucleotide sequences of CHS, DFR and ANS in each dominant species were obtained by sequencing and comparison. Then, to select the most stable reference genes for leaves at different developmental stages and different light conditions, the expression levels of six reference genes, including 18S, Actin, GAPDH, TUB, EF1 and UBQ, were studied by real-time fluorescent quantitative PCR (qRT-PCR), and reference gene stability was analyzed by GeNorm and NormFinder software. The results showed that the expression level of Actin was the most stable in S. superba, A. acuminatissima and C. concinna, and the expression level of GAPDH was the most stable in C. fissa. Finally, the expression levels of the anthocyanin synthesis genes CHS, DFR and ANS were analyzed and found to be consistent with the accumulation trend of anthocyanins in leaves. This study has important theoretical and practical significance for future research into the expression of anthocyanin synthesis-related enzyme genes in the dominant tree species in subtropical forests and reveals that anthocyanin has a photoprotective effect for young leaves in high-light environments.


Assuntos
Antocianinas/biossíntese , Árvores/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Antocianinas/metabolismo , Florestas , Genes de Plantas , Folhas de Planta/genética , Folhas de Planta/metabolismo , RNA de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Alinhamento de Sequência , Análise de Sequência , Árvores/enzimologia , Árvores/metabolismo
16.
Microbiol Res ; 227: 126297, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421711

RESUMO

Many plant growth promoting rhizobacteria such as Bacillus velezensis GJ11 can produce acetoin to trigger induced systemic resistance (ISR) in plants. For improving acetoin production, the mutant strains were respectively constructed by knockout of the gene of bdh (2,3-butanediol dehydrogenase) and gdh (glycerol dehydrogenase) in GJ11, but only GJ11Δbdh produced a high level of acetoin triggering strong ISR against Pseudomonas syringae infection in plants. GJ11Δbdh could induce H2O2 accumulation in plants by producing a high level of acetoin. H2O2 was necessary for triggering ISR against the pathogen infection because after scavenging H2O2 with ascorbic acid or catalase, the inhibition role to pathogen infection induced by acetoin almost disappeared in plants. Further investigation found the plants treated with GJ11Δbdh in an obvious "priming" state, in which the mild immune response was observed such as a slight increase of H2O2 production, callose deposition, and enzymes activity related with defence response (e.g. POD, PAL and PPO). The plants in "priming" could rapidly respond to the pathogen infection accompanying with a significant increase of H2O2 production, callose deposition, and enzymes activity. Collectively, this study provides new insight into the role of acetoin as a strong elicitor of defense response, and ascribes a new approach to construct the mutant strains with high production of acetoin for triggering stronger ISR against pathogens infection in plants.


Assuntos
Acetoína/metabolismo , Arabidopsis/genética , Bacillus/genética , Bacillus/metabolismo , Resistência à Doença/genética , Imunidade Vegetal/genética , Oxirredutases do Álcool/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Ácido Ascórbico/metabolismo , Catalase/metabolismo , Resistência à Doença/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas/genética , Peróxido de Hidrogênio/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/fisiologia , Pseudomonas syringae/patogenicidade , Desidrogenase do Álcool de Açúcar/genética
17.
Acta Crystallogr D Struct Biol ; 75(Pt 8): 733-742, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31373572

RESUMO

p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallography, were exploited to reach these conclusions and provide additional insights.


Assuntos
Oxirredutases do Álcool/química , Mononucleotídeo de Flavina/metabolismo , Ácidos Mandélicos/metabolismo , Oxirredutases do Álcool/genética , Sítios de Ligação , Clonagem Molecular/métodos , Cristalografia por Raios X/métodos , Descarboxilação , Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Oxirredução , Ligação Proteica , Especificidade por Substrato
18.
Genes (Basel) ; 10(8)2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31409011

RESUMO

Hirudin and its variants, as strong inhibitors against thrombin, are present in the saliva of leeches and are recognized as potent anticoagulants. However, their yield is far from the clinical requirement up to now. In this study, the production of hirudin variant 3 (HV3) was successfully realized by cultivating the recombinant Pichia pastoris GS115/pPIC9K-hv3 under the regulation of the promoter of AOX1 encoding alcohol oxidase (AOX). The antithrombin activity in the fermentation broth reached the maximum value of 5000 ATU/mL. To explore an effective strategy for improving HV3 production in the future, we investigated the influence of methanol assimilation on the general gene expression in this recombinant by transcriptomic study. The results showed that methanol was partially oxidized into CO2, and the rest was converted into glycerone-P which subsequently entered into central carbon metabolism, energy metabolism, and amino acid biosynthesis. However, the later metabolic processes were almost all down-regulated. Therefore, we propose that the up-regulated central carbon metabolism, energy, and amino acid metabolism should be beneficial for methanol assimilation, which would accordingly improve the production of HV3.


Assuntos
Hirudinas/genética , Metanol/metabolismo , Pichia/genética , Transcriptoma , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Hirudinas/metabolismo , Pichia/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
19.
Molecules ; 24(14)2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31319487

RESUMO

PpCAD2 was originally isolated from the 'Wangkumbae' pear (Pyrus pyrifolia Nakai), and it encodes for cinnamyl alcohol dehydrogenase (CAD), which is a key enzyme in the lignin biosynthesis pathway. In order to verify the function of PpCAD2, transgenic tomato (Solanum lycopersicum) 'Micro-Tom' plants were generated using over-expression constructs via the agrobacterium-mediated transformation method. The results showed that the PpCAD2 over-expression transgenic tomato plant had a strong growth vigor. Furthermore, these PpCAD2 over-expression transgenic tomato plants contained a higher lignin content and CAD enzymatic activity in the stem, leaf and fruit pericarp tissues, and formed a greater number of vessel elements in the stem and leaf vein, compared to wild type tomato plants. This study clearly indicated that overexpressing PpCAD2 increased the lignin deposition of transgenic tomato plants, and thus validated the function of PpCAD2 in lignin biosynthesis.


Assuntos
Oxirredutases do Álcool/genética , Lignina/genética , Lycopersicon esculentum/genética , Pyrus/genética , Frutas/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/crescimento & desenvolvimento , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento
20.
Int J Biol Macromol ; 138: 781-790, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31351953

RESUMO

To exploit robust biocatalysts for chiral 1-(2-halophenyl)ethanols synthesis, an ortho-haloacetophenones-specific carbonyl reductase (BaSDR1) gene from Bacillus aryabhattai was cloned and expressed in Escherichia coli. The impressive properties regarding BaSDR1 application include preference for NADH as coenzyme, noticeable tolerance against high cosubstrate concentration, and remarkable catalytic performance over a broad pH range from 5.0 to 10.0. The optimal temperature was 35 °C, with a half-life of 3.1 h at 35 °C and 0.75 h at 45 °C, respectively. Notably, BaSDR1 displayed excellent catalytic performance toward various ortho-haloacetophenones, providing chiral 1-(2-halophenyl)ethanols with 99% ee for all the substrates tested. Most importantly, the docking results indicated that the enzyme-substrate interactions and the steric hindrance of halogen atoms act in a push-pull manner in regulating enzyme catalytic ability. These results provide valuable clues for the structure-function relationships of BaSDR1 and the role of halogen groups in catalytic performance, and offer important reference for protein engineering and mining of functional compounds.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Bacillus/genética , Proteínas de Bactérias/genética , Catálise , Clonagem Molecular , Expressão Gênica , Concentração de Íons de Hidrogênio , Íons/química , Metais/química , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA